Typically, purification of intact, non-degraded macromolecules from biological mixtures is difficult due to the presence of lytic enzymes in these mixtures. The presence of lytic enzymes affects the stability of the macromolecule in the biological mixture by causing its degradation. Thus, rapid neutralization of the lytic enzyme at an early step in the purification process is beneficial for increasing the yield and quality (e.g. homogeneity, intactness, and/or functionality) of the purified macromolecule.
The purification is further complicated where the mixture comprising the macromolecule is heterogeneous (i.e. comprises solid particles e.g. particles precipitated in an aqueous solution) and thus cannot be subjected to a purification process in which the mixture is passed at a certain flow rate through a column comprising a packed resin (referred herein as “column purification”) without adding preceding steps e.g. solubilization of the particles, dialysis, filtration and/or the like to obtain a clear solution.
Adding such preceding steps for removing the solid particles from the mixture can be time-consuming and expensive and can also undesirably remove the macromolecule of interest, consequently resulting in a low yield of the purified macromolecule.
For example, purification of fibrinogen in its intact non-degraded form from an aluminum hydroxide precipitate [a byproduct from the manufacture process of factor VIII (FVIII)] is compromised by the presence of a high level of the lytic enzyme plasmin and/or plasminogen present in the precipitate.
U.S. Pat. No. 6,815,535 discloses a method for obtaining a fibrinogen enriched preparation e.g. from heparin precipitated paste, a byproduct from the manufacturing process of FVIII. The method includes adding an effective amount of a sulphated polysaccharide (SPS) to a fibrinogen containing solution to form a fibrinogen containing precipitate; and extracting fibrinogen from the fibrinogen containing precipitate with a solution containing NaCl and ε-aminocaproic acid, a soluble inhibitor of the proteolytic enzyme plasmin/plasminogen.
U.S. Pat. No. 7,125,569 discloses the removal of plasmin/plasminogen from a homogenous/clear mixture using a column packed with immobilized tranexamic acid. U.S. Pat. Nos. 4,341,764 and 4,455,300 disclose different byproduct fractions during the manufacturing of FVIII, e.g. aluminum hydroxide residual fraction, which can be used for the purification of fibronectin and fibrinogen.
There is a need for a fast and effective method for purifying a macromolecule of interest in its intact, non-degraded form from a mixture comprising said macromolecule in the presence of lytic enzymes.